Often, there is a need to perform two or more different assays on the same sample, often in a single vessel and at about the same time. Such assays are known in the art as multiplex or multiplexed assays. Multiplex assays are performed to determine simultaneously the presence/absence or concentration of more than one analyte in the sample being analyzed, or alternatively, several characteristics of a single molecule, such as, the presence of several epitopes on a single protein molecule or identifying alternative alleles of the same gene or nucleic acid sequence.
Detection of nucleotide mutations and polymorphisms is central to the modern science of molecular genetics. For example, allelic discrimination detects different forms of the same gene that differ by nucleotide substitution, insertion, or deletion. In many cases, individuals affected by a given disease display extensive allelic heterogeneity. For example, more than 125 mutations in the human BRCA1 gene have been reported (www.nchgr.nih.gov/dir/lab.sub.—transfer/bic). Mutations in the BRCA1 gene are thought to account for roughly 45% of inherited breast cancer and 80-90% of families with increased risk of early onset breast and ovarian cancer. Other examples of genes for which the population displays extensive allelic heterogeneity and which have been implicated in disease include CFTR (cystic fibrosis), dystrophin (Duchenne muscular dystrophy, and Becker muscular dystrophy), and p53 (Li-Fraumeni syndrome) among many others.
Accuracy in detection of mutations is extremely important, particularly in clinical settings. Methods for mutation detection can be divided into two groups: scanning methods that can detect previously unknown nucleotide differences, and methods designed to detect specific, known mutations or polymorphisms. Methods for the detection of known nucleotide differences currently include the following techniques: hybridization with allele-specific oligonucleotides (ASO); allele-specific PCR; solid-phase minisequencing; oligonucleotide ligation assay; allele-specific ligase chain reaction (LCR) among others. For the analysis of genomic DNA, these methods involve amplification of a specific DNA segment, followed by detection analysis to determine which allele is present. These methods are, however, ill suited for automated analysis of multiple mutations or multiple samples.
An automated method for detecting mutations, called “spectral genotyping,” has been described previously, in which alleles are identified by fluorescent colors generated in sealed amplification tubes. In this technique, amplification is carried out in the presence of molecular beacons, which are probes that become fluorescent when they hybridize to their target. Tyagi et al. demonstrated that probes with a reporter at the 5′ end and a quencher at the 3′ end can be used to distinguish alleles. Tyagi S and Kramer F R (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303-308. Molecular beacons are hairpin-shaped, single-stranded oligonucleotides consisting of a probe sequence embedded within complementary sequences that form a hairpin stem. A fluorophore is covalently attached to one end of the oligonucleotide, and a non-fluorescent quencher is covalently attached to the other end. In the absence of a target, the fluorophore is held close to the quencher and fluorescence cannot occur. When the probe binds to its target, the rigidity of the probe-target helix forces the stem to unwind, resulting in the separation of the fluorophore and quencher, and restoration of fluorescence. These probes can detect a number of different targets in the same solution. This is accomplished by constructing a different molecular beacon for each target and attaching a differently colored fluorophore to each. The probes are placed in the same amplification tube, and the color that develops indicates which targets were present. For genotyping alleles, two molecular beacons are used, one specific for the wild-type allele and labeled with a green fluorophore and the other specific for the mutant allele and labeled with a red fluorophore. The appearance of green fluorescence during amplification indicates homozygous wild-types, red fluorescence indicates homozygous mutants, and both green and red fluorescence indicates heterozygotes.
This procedure was used in the past to distinguish, for example, alleles of the beta-chemokine receptor 5 (CCR5) gene that determines susceptibility to infection by the human immunodeficiency virus (HIV). Individuals homozygous for a 32-nucleotide deletion in this gene (CCR5D32) are largely resistant to HIV infection, despite multiple sexual contacts with HIV-infected individuals, and heterozygotes are partially protected against disease progression. To understand the susceptibility of human populations to the spread of HIV, large-scale epidemiological studies of the distribution of this mutant allele are needed, necessitating high-throughput assays. Therefore, an automated spectral genotyping assay was developed that identifies CCR5 alleles. For the detection of the wild-type allele, a fluorescein-labeled molecular beacon was prepared whose probe sequence was complementary to the region that is deleted in the mutant; for the detection of the mutant allele, a tetramethylrhodamine-labeled molecular beacon was synthesized that was complementary to the sequences flanking the region of the deletion, which are brought together in the mutant. Human DNA samples were used as templates for polymerase chain reactions (PCRS) in which the region of the CCR5 gene that encompasses the site of the D32 mutation was amplified in the presence of both molecular beacons. The sequence of the wild-type-specific molecular beacon was green fluorescent fluorescein-5′-CGGTCTGGAAATTCTTCCAGAATTGATACTGACCGG-3′-DABCYL and the sequence of the mutant-specific molecular beacon was red fluorescent tetramethylrhodamine-5′-CGGCTATCTTTAATGTATGGAAAATGAGAGCCG-3-DABCYL, and where DABCYL is the quencher 4-(4′-dimethylaminophenylazo) benzoic acid. Furthermore, the allele discrimination was demonstrated for two alleles in the human insulin gene that differ by only a single A-T nucleotide substitution.
It is apparent from the above that one is limited to only three reaction outcomes at one time. Thus, while assays for allele identification are now available, these assays can not measure more than a few distinct parameters or analytes simultaneously. This limitation stems from the technical difficulty of measuring several labels simultaneously.
This problem with conventional multiplex assays has been recently solved with Luminex proprietary LabMAP system which typically detects 100 analytes simultaneously in the same reagent mixture (see for detailed description of the technology at website www.luminexcorp.com). This significant advantage is largely attributable to the availability of fluorescently addressable microspheres, specially designed flow cytometry apparatus and related assay methods as described in detail in commonly owned U.S. Pat. Nos. 6,046,807; 5,981,180; and 5,736,330. The present invention provides further and unexpected improvements to this multiplex method and allows the identification of more than 100 different analytes simultaneously, while using the same set of 100 fluorescently addressable microspheres.